Abstract BackgroundPreoperative skeletal muscle deficiency is an established risk factor for poor survival outcomes in patients with renal cell carcinoma (RCC). However, given the dynamic nature of skeletal muscle associated with malignancy, there is a need to evaluate the prognostic benefit of muscle area change from the preoperative to postoperative period. We hypothesize that an improvement in muscle area following nephrectomy, measured by linear segmentation of L3 psoas and paraspinal musculature, is associated with improvement in overall survival (OS) and cancer specific survival (CSS) for patients with pT3 and pT4 RCC.MethodsWe retrospectively analysed 270 pT3 and pT4 RCC patients who underwent nephrectomy from March 2004 to February 2020 with available preoperative and postoperative axial CT or MRI studies segmented at the L3 vertebrae. The majority were N0 (79%) and M0 (68%). Psoas and paraspinal muscles were measured bilaterally using a validated digital ruler tool. Total muscle area (TMA) was calculated by aggregating the area of all four muscles and total muscle area index (TMI) by dividing the TMA by height squared (m2). The prognostic value of postoperative muscle improvement, defined as any increase in muscle area index, was analysed using Kaplan–Meier and Cox proportional stepwise hazard models.ResultsMedian time between preoperative scans and surgery was approximately 22 days and between surgery and postoperative scans 172 days. One hundred twenty‐one patients (44.8%) had an increase in total muscle area index post‐nephrectomy (IQR = 33.4; P ≤ 0.0001). On Kaplan–Meier analysis, postoperative improvement in TMI was associated with decreased odds of mortality (P = 0.0024) with a median follow‐up of 38.6 months. In a multivariable Cox regression analysis, improvement of TMI was associated with increased OS (HR = 0.52, 95% CI 0.35–0.78, P < 0.001) and increased CSS (HR = 0.55, 95% CI 0.32–0.94, P = 0.030). A 5% or more improvement in TMI was also associated with increased OS (HR = 0.53, 95% CI 0.34–0.84, P = 0.006) and increased CSS (HR = 0.46, 95% CI 0.24–0.86, P = 0.015).ConclusionsAny improvement in TMI between preoperative and initial postoperative imaging after nephrectomy was associated with increased OS and CSS in patients with pT3 and pT4 RCC. Perioperative linear segmentation is an efficient tool that may improve current prognostication methods and can be performed on any imaging software platform.

Abstract BackgroundPeak oxygen uptake (peak VO2) is known not only as an index of aerobic fitness but also one of an index of life expectancy. Frailty, sarcopenia, and cachexia are associated with a poor prognosis and high mortality. The purpose of this study was to determine the relationships of peak VO2 with the features of sarcopenia, frailty, and cachexia, to provide insight into which might mediate the poor prognosis.MethodsThe first group of participants was 175 community‐dwelling older Japanese outpatients (58 men and 117 women; mean age 77.6 ± 6.4 years), in whom we assessed the features of sarcopenia, frailty, and cachexia, and measured peak VO2 during cardiopulmonary exercise. To confirm the relationships, we analysed another group of 162 participants (77.3 ± 5.5 years).ResultsThere were significant correlations between peak VO2 and the features of sarcopenia, frailty, and cachexia, with the exception of high sensitivity C‐reactive protein. Multiple linear regression analysis for the prediction of peak VO2 (mL/min) identified following formula: predicted peak VO2 = −11.6 × age (years) + 25.5 × haemoglobin concentration (g/dL) + 114.2 × skeletal muscle mass index (kg/m2) + 8.9 × hand grip strength (kg) + 226.4 × usual walking speed (m/s) − 65.8 × fatiguability (absence 0, presence 1) − 177.4 × chronic heart failure (absence 0, presence 1) + 437.1 (R2 = 0.627, P < 0.001). The validity of the formula was confirmed with another group (r = 0.78, P < 0.001).ConclusionsThis study has identified the features of sarcopenia, frailty, and cachexia that are related to peak VO2 in an older population.

Eccentric contractions induce muscle damage (EIMD) that compromises muscle function. Poor recovery from EIMD has been suggested to be a contributor to the decline in muscle function evident in sarcopenia, but it is unclear which aspects of muscle function are more susceptible to disruption by EIMD in old versus young muscle. The purpose of this study was to determine the extent of impairment in contractile function (force, fatigue, tetanus and twitch kinetics) during the recovery from EIMD in very old (VO) mice compared to young adult (YA). Male CB6F1 were obtained from National Institure of Aging colony. VO mice were 29-31 months of age, and YA mice were 7-9 months of age. The plantarflexor muscles were subjected to 20 eccentric contractions in vivo to induce injury (EIMD). Changes in tetanic force and kinetics were assessed before EIMD, immediately after EIMD and 3 days after EIMD (3dEIMD). Force-frequency and rates of fatigue were assessed 3d-EIMD and compared with baseline. Histological analysis was conducted in injured and non-injured contralateral gastrocnemius muscle. There was a greater loss in isometric tetanic force immediately following EIMD in VO compared with YA (-31.6% ± 10.4 vs. -21.7% ± 6.0, P < 0.05). At 3d-EIMD, the rate of contraction of tetanus began to recover in VO, but not in YA (20.8% vs. -6.8%, P < 0.05), whereas the extent of recovery of force tended to be greater in VO than YA (39.3% vs. 17.1%, P = 0.08) when compared with tetanic function immediately after injury. Compared with function pre-injury (baseline), VO and YA had similar deficits in tetanic force (-7.3% ± 5.3 vs. -9.2% ± 6.0, respectively) and kinetics at Day 3. Twitch kinetics (rate of relaxation) recovered faster in VO compared with YA. The rate of muscle fatigue was similar to baseline values, with VO continuing to be more fatigue resistant than YA 3d-EIMD. There were no detectable differences in muscle mass or myofibre cross-sectional area despite continued deficits in force following EIMD in either age group. Despite clear functional deficits and greater susceptibility to injury, aged sarcopenic muscle exhibited a similar ability to recover contractile function to younger muscle following EIMD. In addition, neither age group showed accelerated muscle fatigue in the recovery phase after EIMD; thus, sarcopenic mouse muscles do not appear to be more susceptible to long-term functional impairment than young healthy muscles.

Abstract BackgroundCachexia, a complex multi‐organ syndrome, shortens survival time of patients, particularly those with cancer. Many studies and clinical trials have been carried out to identify cachexia‐inducing factors and potential treatments for cancer cachexia over the last 20 years. Of these factors, some are promising targets for treatment in humans, owing to their expression profiles in patients. Several clinical interventions, which act on either cachexia‐inducing factors or tissues affected by cachexia, have been developed. Some have had positive effects in the treatment of cancer cachexia; however, the question remains whether these interventions reverse cancer cachexia and could be used as standard interventions for disease treatment. The aim of this review is to understand the basic mechanisms and factors that induce cancer cachexia and their efficacies in clinical trials, providing a better outlook for future studies of cancer cachexia.MethodsA systematic search was performed using PubMed and ClinicalTrials.gov databases for cachexia mediators and clinical trials.ResultsOf all databases and peer‐reviewed facts considered, 256 papers and 35 clinical trials were included in this systematic review. Twenty‐one mediators were identified, and 17 clinical interventions were reported in these studies. Outcomes of these clinical trials were assessed on changes in overall survival, body weight, lean body mass, appetite, muscle strength, muscle function, quality of life, and cytokine levels.ConclusionsThere is no current standard or successful intervention for treating cancer cachexia. Further research is needed to improve our understanding of initiators of cachexia to achieve successful outcomes in cachexia clinical trials.

Abstract BackgroundLoss of skeletal muscle volume and functional limitations are poor prognostic markers in breast cancer patients. Several molecular defects in skeletal muscle including reduced myoblast determination protein 1 (MyoD) levels and increased protein turn over due to enhanced proteosomal activity have been suggested as causes of skeletal muscle loss in cancer patients. However, it is unknown whether molecular defects in skeletal muscle are dependent on tumour aetiology.MethodsWe characterized functional and molecular defects of skeletal muscle in mouse mammary tumour virus (MMTV)‐Neu (Neu+) mice (n = 6–12), an animal model that represents HER2 + human breast cancer, and compared the results with well‐characterized luminal B breast cancer model MMTV‐PyMT (PyMT+). Functional studies such as grip strength, rotarod performance, and ex vivo muscle contraction were performed to measure the effects of cancer on skeletal muscle. Expression of muscle‐enriched genes and microRNAs as well as circulating cytokines/chemokines were measured. Because nuclear factor‐kappaB (NF‐κB) pathway plays a significant role in skeletal muscle defects, the ability of NF‐κB inhibitor dimethylaminoparthenolide (DMAPT) to reverse skeletal muscle defects was examined.ResultsNeu+ mice showed skeletal muscle defects similar to accelerated aging. Compared with age and sex‐matched wild type mice, Neu+ tumour‐bearing mice had lower grip strength (202 ± 6.9 vs. 179 ± 6.8 g grip force, P = 0.0069) and impaired rotarod performance (108 ± 12.1 vs. 30 ± 3.9 s, P &lt; 0.0001), which was consistent with reduced muscle contractibility (P &lt; 0.0001). Skeletal muscle of Neu+ mice (n = 6) contained lower levels of CD82 + (16.2 ± 2.9 vs. 9.0 ± 1.6) and CD54 + (3.8 ± 0.5 vs. 2.4 ± 0.4) muscle stem and progenitor cells (P &lt; 0.05), suggesting impaired capacity of muscle regeneration, which was accompanied by decreased MyoD, p53, and miR‐486 expression in muscles (P &lt; 0.05). Unlike PyMT+ mice, which showed skeletal muscle mitochondrial defects including reduced mitochondria levels and peroxisome proliferator‐activated receptor gamma co‐activator 1 (beta), Neu + mice displayed accelerated aging‐associated changes including muscle fibre shrinkage and increased extracellular matrix deposition. Circulating ‘aging factor’ and cachexia and fibromyalgia‐associated chemokine C‐C motif chemokine ligand 11 (Ccl11) was elevated in Neu+ mice (1439.56 ± 514 vs. 1950 ± 345 pg/mL, P &lt; 0.05). Treatment of Neu+ mice with DMAPT significantly restored grip strength (205 ± 6 g force), rotarod performance (74 ± 8.5 s), reversed molecular alterations associated with skeletal muscle aging, reduced circulating Ccl11 (1083.26 ± 478 pg/mL), and improved animal survival.ConclusionsThese results suggest that breast cancer subtype has a specific impact on the type of molecular and structural changes in skeletal muscle, which needs to be taken into consideration while designing therapies to reduce breast cancer‐induced skeletal muscle loss and functional limitations.

Cachexia, a syndrome of muscle atrophy, adipose loss, and anorexia, is associated with reduced survival in cancer patients. The colon adenocarcinoma C26c20 cell line secretes the cytokine leukemia inhibitory factor (LIF) which induces cachexia. We characterized how LIF promotes cachexia-associated weight loss and anorexia in mice through JAK-dependent changes in adipose and hypothalamic tissues. Cachexia was induced in vivo with the heterotopic allotransplanted administration of C26c20 colon adenocarcinoma cells or the intraperitoneal administration of recombinant LIF in the absence or presence of JAK inhibitors. Blood, adipose, and hypothalamic tissues were collected and processed for cyto/adipokine ELISAs, immunoblot analysis, and quantitative RT-PCR. Cachexia-associated lipolysis was induced in vitro by stimulating differentiated adipocytes with recombinant LIF or IL-6 in the absence or presence of lipase or JAK inhibitors. These adipocytes were processed for glycerol release into the media, immunoblot analysis, and RT-PCR. Tumor-secreted LIF induced changes in adipose tissue expression and serum levels of IL-6 and leptin in a JAK-dependent manner influencing cachexia-associated adipose wasting and anorexia. We identified two JAK inhibitors that block IL-6 family-mediated adipocyte lipolysis and IL-6 induction using an in vitro cachexia lipolysis assay. JAK inhibitors administered to the in vivo C26c20 cancer cachexia mouse models led to 1) a decrease in STAT3 phosphorylation in hypothalamic and adipose tissues, 2) a reverse in the cachexia serum cyto/adipokine signature, 3) a delay in cancer cachexia-associated anorexia and adipose loss, and 4) an improvement in overall survival. JAK inhibitors suppress LIF-associated adipose loss and anorexia in both in vitro and in vivo models of cancer cachexia.

Cancer cachexia is a multifactorial wasting syndrome that is characterized by the loss of skeletal muscle mass and weakness, which compromises physical function, reduces quality of life, and ultimately can lead to mortality. Experimental models of cancer cachexia have recapitulated this skeletal muscle atrophy and consequent decline in muscle force generating capacity. We address these issues in a novel transgenic mouse model Kras, Trp53 and Pdx-1-Cre (KPC) of pancreatic ductal adenocarcinoma (PDA) using multi-parametric magnetic resonance (mp-MR) measures. KPC mice (n = 10) were divided equally into two groups (n = 5/group) depending on the size of the tumor i.e. tumor size <250 mm3 and >250 mm3. Using mp-MR measures, we demonstrated the changes in the gastrocnemius muscle at the microstructural level. In addition, we evaluated skeletal muscle contractile function in KPC mice using an in vivo approach. Increase in tumor size resulted in decrease in gastrocnemius maximum cross sectional area, decrease in T2 relaxation time, increase in magnetization transfer ratio, decrease in mean diffusivity, and decrease in radial diffusivity of water across the muscle fibers. Finally, we detected significant decrease in absolute and specific force production of gastrocnemius muscle with increase in tumor size. Our findings indicate that increase in tumor size may cause alterations in structural and functional parameters of skeletal muscles and that MR parameters may be used as sensitive biomarkers to noninvasively detect structural changes in cachectic muscles.

Muscle wasting is a debilitating co-morbidity affecting most advanced cancer patients. Alongside enhanced muscle catabolism, defects in muscle repair/regeneration contribute to cancer-associated wasting. Among the factors implicated in suppression of muscle regeneration are cytokines that interfere with myogenic signal transduction pathways. Less understood is how other cancer/wasting-associated cues, such as metabolites, contribute to muscle dysfunction. This study investigates how the metabolite succinate affects myogenesis and muscle regeneration. We leveraged an established ectopic metabolite treatment (cell permeable dimethyl-succinate) strategy to evaluate the ability of intracellular succinate elevation to 1) affect myoblast homeostasis (proliferation, apoptosis), 2) disrupt protein dynamics and induce wasting-associated atrophy, and 3) modulate in vitro myogenesis. In vivo succinate supplementation experiments (2% succinate, 1% sucrose vehicle) were used to corroborate and extend in vitro observations. Metabolic profiling and functional metabolic studies were then performed to investigate the impact of succinate elevation on mitochondria function. We found that in vitro succinate supplementation elevated intracellular succinate about 2-fold, and did not have an impact on proliferation or apoptosis of C2C12 myoblasts. Elevated succinate had minor effects on protein homeostasis (~25% decrease in protein synthesis assessed by OPP staining), and no significant effect on myotube atrophy. Succinate elevation interfered with in vitro myoblast differentiation, characterized by significant decreases in late markers of myogenesis and fewer nuclei per myosin heavy chain positive structure (assessed by immunofluorescence staining). While mice orally administered succinate did not exhibit changes in overall body composition or whole muscle weights, these mice displayed smaller muscle myofiber diameters (~6% decrease in the mean of non-linear regression curves fit to the histograms of minimum feret diameter distribution), which was exacerbated when muscle regeneration was induced with barium chloride injury. Significant decreases in the mean of non-linear regression curves fit to the histograms of minimum feret diameter distributions were observed 7 days and 28 days post injury. Elevated numbers of myogenin positive cells (3-fold increase) supportive of the differentiation defects observed in vitro were observed 28 days post injury. Metabolic profiling and functional metabolic assessment of myoblasts revealed that succinate elevation caused both widespread metabolic changes and significantly lowered maximal cellular respiration (~35% decrease). This study broadens the repertoire of wasting-associated factors that can directly modulate muscle progenitor cell function and strengthens the hypothesis that metabolic derangements are significant contributors to impaired muscle regeneration, an important aspect of cancer-associated muscle wasting.

Abstract BackgroundMuscle regenerative capacity reduces during aging. Decline in the muscle stem cell (MuSC) pool is thought to be a cause of age‐related muscle regeneration insufficiency. However, why the number of MuSCs decreases with age remains unclear. We previously found that the calcitonin receptor (Calcr) is expressed by MuSCs and plays a pivotal role in maintaining the MuSC pool. In this study, we examined whether Calcr is involved in the age‐related decrease in MuSC number.Methods and resultsCalcr expression in MuSCs was assessed by immunofluorescence staining using muscle sections during postnatal growth and during aging. The role of Calcr during development, postnatal growth and adult stage was examined using mice conditionally deleted Calcr in myogenic lineage cells. To explore the significance of Calcr signaling on age‐related changes in MuSC number, expression levels of endogenous Calcr ligands were examined. Immunohistological examination revealed that Calcr expression grew stronger as MuSCs became quiescent. Conditional Calcr deletion in early myogenic lineage cells did not affect the initial capacity of the MuSC pool, but promoted age‐dependent MuSC loss. Expression of endogenous Calcr ligands remained unchanged with aging. Calcr expression in MuSCs, however, decreased significantly in aged muscle compared with young muscle in both mice and humans, and this reduction was accompanied by a loss of MuSCs.ConclusionsOur study revealed significant reduction of Calcr in MuSCs with age, possibly leading to age‐related loss of the MuSC pool. Thus, Calcr signaling could represent a promising target for preventing aging‐associated muscle regeneration insufficiency.